OECD/OCDE 451

Adopted:
25 June 2018

OECD GUIDELINE FOR THE TESTING OF CHEMICALS:

CARCINOGENICITY STUDIES

INTRODUCTION

1. OECD Guidelines for the Testing of Chemicals (TGs) are periodically reviewed in the light of
scientific progress, changing assessment practices and animal welfare considerations. The original Test
Guideline 451 on Carcinogenicity Studies was adopted in 1981. Development of a revised TG 451 was
considered necessary, in order to reflect recent developments in the field of animal welfare and
regulatory requirements (1) (2) (3) (4) (5). The updating of TG 451 has been carried out in parallel with
revisions of the Test Guidelines 452, Chronic Toxicity Studies, and 453, Combined Chronic
Toxicity\Carcinogenicity Studies, and with the objective of obtaining additional information from the
animals used in the study and providing further detail on dose selection. This Test Guideline is designed
to be used in the testing of a broad range of chemicals, including pesticides and industrial chemicals. It
should be noted however that some details and requirements may differ for pharmaceuticals (see
International Conference on Harmonisation (ICH) Guidance S1B on Testing for Carcinogenicity of
Pharmaceuticals).

2. The majority of carcinogenicity studies are carried out in rodent species, and this Test
Guideline is intended therefore to apply primarily to studies carried out in these species. Should such
studies be required in non-rodent species, the principles and procedures outlined in this Guideline
together with those outlined in OECD TG 409, Repeated Dose 90-day Oral Toxicity Study in
Non-Rodents (6) should be applied, with appropriate modifications. Further guidance is available in the
OECD Guidance Document No. 116 on the Design and Conduct of Chronic Toxicity and
Carcinogenicity Studies (7).

3. The three main routes of administration used in carcinogenicity studies are oral, dermal and
inhalation. The choice of the route of administration depends on the physical and chemical characteristics
of the test chemical and the predominant route of exposure of humans. Additional information on choice
of route of exposure is provided in Guidance Document No. 116 (7).

4. This Guideline focuses on exposure via the oral route, the route most commonly used in
carcinogenicity studies. While carcinogenicity studies involving exposure via the dermal or inhalation
routes may also be necessary for human health risk assessment and/or may be required under certain
regulatory regimes, both routes of exposure involve considerable technical complexity. Such studies will
need to be designed on a case-by-case basis, although the Guideline outlined here for the assessment and
© OECD, (2018)

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In accordance with the Decision of the Council on a Delegation of Authority to amend Annex I of the Decision of the Council on the Mutual
Acceptance of Data in the Assessment of Chemicals [C(2018)49], this Guideline was amended by the OECD’s Joint Meeting of the Chemicals
Committee and the Working Party on Chemicals, Pesticides and Biotechnology by written procedure on 25 June 2018.
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evaluation of carcinogenicity by oral administration could form the basis of a protocol for inhalation
and/or dermal studies, with respect to recommendations for treatment periods, clinical and pathology
parameters, etc. OECD Guidance is available on the administration of test chemicals by the dermal (7),
and inhalation routes (7) (8). TG 412 (9) and TG 413 (10), together with the associated OECD Guidance
Document on acute inhalation testing (8), should be specifically consulted in the design of longer term
studies involving exposure via the inhalation route. TG 410 (11) should be consulted in the case of
testing carried out by the dermal route.

5. The carcinogenicity study provides information on the possible health hazards likely to arise
from repeated exposure for a period lasting up to the entire lifespan of the species used. The study will
provide information on the toxic effects of the substance including potential carcinogenicity, and may
indicate target organs and the possibility of accumulation. It can provide an estimate of the
no-observed-adverse effect level for toxic effects and, in the case of non-genotoxic carcinogens, for
tumour responses, which can be used for establishing safety criteria for human exposure. The need for
careful clinical observations of the animals, so as to obtain as much information as possible, is also
stressed.

6. The objectives of carcinogenicity studies covered by this test guideline include:

 The identification of the carcinogenic properties of a chemical, resulting in an increased
incidence of neoplasms, increased proportion of malignant neoplasms or a reduction in the time
to appearance of neoplasms, compared with concurrent control groups,
 The identification of target organ(s) of carcinogenicity;
 The identification of the time to appearance of neoplasms;
 Characterisation of the tumour dose-response relationship;
 Identification of a no-observed-adverse-effect level (NOAEL) or point of departure for
establishment of a Benchmark Dose (BMD);
 Extrapolation of carcinogenic effects to low dose human exposure levels;
 Provision of data to test hypotheses regarding mode of action (2) (7) (12) (13) (14) (15).

INITIAL CONSIDERATIONS

7. In the assessment and evaluation of the potential carcinogenicity of a chemical, all available
information on the test chemical should be considered by the testing laboratory prior to conducting the
study, in order to focus the design of the study to more efficiently test for carcinogenic potential and to
minimize animal usage. Information on, and consideration of, the mode of action of a suspected
carcinogen (2) (7) (12) (13) (14) (15) is particularly important, since the optimal design may differ
depending on whether the substance is a known or suspected genotoxic carcinogen. Further guidance on
mode of action considerations can be found in Guidance Document No.116 (7).

8. Information that will assist in the study design includes the identity, chemical structure, and
physico-chemical properties of the test chemical; results of any in vitro or in vivo toxicity tests including
genotoxicity tests; anticipated use(s) and potential for human exposure; available (Q)SAR data,
mutagenicity/genotoxicity, carcinogenicity and other toxicological data on structurally-related
substances; available toxicokinetic data (single dose and also repeat dose kinetics where available) and
data derived from other repeated exposure studies. Assessment of carcinogenicity should be carried out
after initial information on toxicity has been obtained from repeated dose 28-day and/or 90-day toxicity
tests. Short-term cancer initiation-promotion tests could also provide useful information. A phased

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testing approach to carcinogenicity testing should be considered as part of the overall assessment of the
potential adverse health effects of a particular chemical (16) (17) (18) (19).

9. The statistical methods most appropriate for the analysis of results, given the experimental
design and objectives, should be established before commencing the study. Issues to consider include
whether the statistics should include adjustment for survival, analysis of cumulative tumour risks relative
to survival duration, analysis of the time to tumour and analysis in the event of premature termination of
one or more groups. Guidance on the appropriate statistical analyses and key references to internationally
accepted statistical methods are given in Guidance Document No.116 (7), and also in Guidance
Document No. 35 on the analysis and evaluation of chronic toxicity and carcinogenicity studies (20).

10. In conducting a carcinogenicity study, the guiding principles and considerations outlined in the
OECD Guidance Document No. 19 on the recognition, assessment, and use of clinical signs as humane
endpoints for experimental animals used in safety evaluation (21), in particular paragraph 62 thereof,
should always be followed. This paragraph states that “In studies involving repeated dosing, when an
animal shows clinical signs that are progressive, leading to further deterioration in condition, an
informed decision as to whether or not to humanely kill the animal should be made. The decision should
include consideration as to the value of the information to be gained from the continued maintenance of
that animal on study relative to its overall condition. If a decision is made to leave the animal on test, the
frequency of observations should be increased, as needed. It may also be possible, without adversely
affecting the purpose of the test, to temporarily stop dosing if it will relieve the pain or distress, or
reduce the test dose.”

11. Detailed guidance on and discussion of the principles of dose selection for chronic toxicity and
carcinogenicity studies can be found in Guidance Document No.116 (7) as well as two International Life
Sciences Institute publications (22) (23). The core dose selection strategy is dependent on the primary
objective or objectives of the study (paragraph 6). In selecting appropriate dose levels, a balance should
be achieved between hazard screening on the one hand and characterization of low-dose responses and
their relevance on the other. This is particularly relevant in the situation where a combined chronic
toxicity and carcinogenicity study (TG 453) is to be carried out (paragraph 12).

12. Consideration should be given to carrying out a combined chronic toxicity and carcinogenicity
study (TG 453), rather than separate execution of a chronic toxicity study (TG 452) and carcinogenicity
study (TG 451). The combined test provides greater efficiency in terms of time and cost compared to
conducting two separate studies, without compromising the quality of the data in either the chronic phase
or the carcinogenicity phase. Careful consideration should however be given to the principles of dose
selection (paragraphs 11 and 22-25) when undertaking a combined chronic toxicity and carcinogenicity
study (TG 453), and it is also recognised that separate studies may be required under certain regulatory
frameworks.

13. Definitions used in the context of this Test Guideline can be found in Guidance Document No.
116 (7).

PRINCIPLE OF THE TEST

14. The test chemical is administered daily in graduated doses to several groups of test animals for
the majority of their life span, normally by the oral route. Testing by the inhalation or dermal route may
also be appropriate. The animals are observed closely for signs of toxicity and for the development of
neoplastic lesions. Animals which die or are killed during the test are necropsied and, at the conclusion
of the test, surviving animals are killed and necropsied.

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DESCRIPTION OF METHOD

Selection of animal species

15. This Guideline primarily covers assessment and evaluation of carcinogenicity in rodents
(paragraph 2). The use of non-rodent species may be considered when available data suggest that they
are more relevant for the prediction of health effects in humans. The choice of species should be
justified. The preferred rodent species is the rat, although other rodent species, e.g., the mouse, may be
used. Although the use of the mouse in carcinogenicity testing may have limited utility (24) (25) (26),
under some current regulatory programmes carcinogenicity testing in the mouse is still required. Rats and
mice have been preferred experimental models because of their relatively short life span, their
widespread use in pharmacological and toxicological studies, their susceptibility to tumour induction,
and the availability of sufficiently characterised strains. As a consequence of these characteristics, a large
amount of information is available on their physiology and pathology. Additional information on choice
of species and strain is provided in Guidance Document No.116 (7).

16. Young healthy adult animals of commonly used laboratory strains should be employed. The
carcinogenicity study should preferably be carried out in animals from the same strain and source as
those used in preliminary toxicity study(ies) of shorter duration although, if animals from this strain and
source are known to present problems in achieving the normally accepted criteria of survival for long-
term studies (see Guidance Document No. 116 (7)), consideration should be given to using a strain of
animal that has an acceptable survival rate for the long-term study. The females should be nulliparous
and non-pregnant.

Housing and feeding

17. Animals may be housed individually, or be caged in small groups of the same sex; individual
housing should be considered only if scientifically justified (27) (28) (29). Cages should be arranged in
such a way that possible effects due to cage placement are minimised. The temperature in the
experimental animal room should be 22C (± 3C). Although the relative humidity should be at least
30% and preferably not exceed 70% other than during room cleaning, the aim should be 50-60%.
Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional
laboratory diets may be used with an unlimited supply of drinking water. The diet should meet all the
nutritional requirements of the species tested and the content of dietary contaminants, including but not
limited to pesticide residues, persistent organic pollutants, phytoestrogens, heavy metals and mycotoxins,
that might influence the outcome of the test, should be as low as possible. Analytical information on the
nutrient and dietary contaminant levels should be generated periodically, at least at the beginning of the
study and when there is a change in the batch used, and should be included in the final report. Analytical
information on the drinking water used in the study should similarly be provided. The choice of diet may
be influenced by the need to ensure a suitable admixture of a test chemical and to meet the nutritional
requirements of the animals when the test chemical is administered by the dietary route.

Preparation of animals
18. Healthy animals, which have been acclimated to laboratory conditions for at least 7 days and
have not been subjected to previous experimental procedures, should be used. In the case of rodents,
dosing of the animals should begin as soon as possible after weaning and acclimatisation and preferably
before the animals are 8 weeks old. The test animals should be characterised as to species, strain, source,
sex, weight and age. At the commencement of the study, the weight variation for each sex of animal used
should be minimal and not exceed ± 20 % of the mean weight of all the animals within the study,
separately for each sex. Animals should be randomly assigned to the control and treatment groups. After

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randomisation, there should be no significant differences in mean body weights between groups within
each sex. If there are statistically significant differences, then the randomisation step should be repeated,
if possible. Each animal should be assigned a unique identification number, and permanently marked
with this number by tattooing, microchip implant, or other suitable method.

PROCEDURE

Number and sex of animals

19. Both sexes should be used. A sufficient number of animals should be used so that a thorough
biological and statistical evaluation is possible. Each dose group and concurrent control group should
therefore contain at least 50 animals of each sex. Depending on the aim of the study, it may be possible
to increase the statistical power of the key estimates by differentially allocating animals unequally to the
various dose groups, with more than 50 animals in the low dose groups; e.g., to estimate the carcinogenic
potential at low doses. However it should be recognized that a moderate increase in group size will
provide relatively little increase in statistical power of the study. Further information on statistical design
of the study and choice of dose levels to maximise statistical power is provided in Guidance Document
No. 116 (7).

Provision for interim kills and satellite (sentinel) groups

20. The study may make provision for interim kills, e.g., at 12 months, to provide information on
progression of neoplastic changes and mechanistic information, if scientifically justified. Where such
information is already available from previous repeat dose toxicity studies on the substance, interim kills
may not be scientifically justified. If interim kills are included in the study design, the number of
animals in each dose group scheduled for an interim kill will normally be 10 animals per sex, and the
total number of animals included in the study design should be increased by the number of animals
scheduled to be killed before the completion of the study. An additional group of sentinel animals
(typically 5 animals per sex) may be included for monitoring of disease status, if necessary, during the
study (30). Further guidance is provided in Guidance Document No. 116 (7).

Dose groups and dosage

21. Guidance on all aspects of dose selection and dose level spacing is provided in Guidance
Document No. 116 (7). At least three dose levels and a concurrent control should be used. Dose levels
will generally be based on the results of shorter-term repeated dose or range finding studies and should
take into account any existing toxicological and toxicokinetic data available for the test chemical or
related materials.

22. In the dose selection the investigator should also consider and ensure that data generated is
adequate to fulfil the regulatory requirements across OECD countries as appropriate (e.g., hazard and
risk assessment, classification and labelling, ED assessment, etc.)

23. Unless limited by the physical-chemical nature or biological effects of the test chemical, the
highest dose level should be chosen to identify the principal target organs and toxic effects while
avoiding suffering, severe toxicity, morbidity, or death. While taking into account the factors outlined in
paragraph 23 below, the highest dose level should normally be chosen to elicit evidence of toxicity, as
evidenced by, for example, depression of body weight gain (approximately 10%). However, dependent
on the objectives of the study (see paragraph 6), a top dose lower than the dose providing evidence of
toxicity may be chosen, e.g., if a dose elicits an adverse effect of concern that nonetheless has little
impact on lifespan or body weight.

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24. Dose levels and dose level spacing may be selected to establish a dose-response and, depending
on the mode of action of the test chemical, a NOAEL or other intended outcome of the study, e.g. a BMD
(see paragraph 25) at the lowest dose level. Factors that should be considered in the placement of lower
doses include the expected slope of the dose–response curve, the doses at which important changes may
occur in metabolism or mode of toxic action, where a threshold is expected, or where a point of departure
for low-dose extrapolation is expected.

25. The dose level spacing selected will depend on the characteristics of the test chemical, and
cannot be prescribed in this Guideline, but two to four fold intervals frequently provide good test
performance for setting the descending dose levels and addition of a fourth test group is often preferable
to using very large intervals (e.g., more than a factor of about 6-10) between dosages. In general, the use
of factors greater than 10 should be avoided, and should be justified if used.

26. As discussed further in Guidance Document No. 116 (7), points to be considered in dose
selection include:

 Known or suspected nonlinearities or inflection points in the dose–response;

 Toxicokinetics, and dose ranges where metabolic induction, saturation, or nonlinearity

between external and internal doses does or does not occur;

 Precursor lesions, markers of effect, or indicators of the operation of key underlying

biological processes;

 Key (or suspected) aspects of mode of action, such as doses at which cytotoxicity begins
to arise, hormone levels are perturbed, homeostatic mechanisms are overwhelmed, etc.;

 Regions of the dose–response curve where particularly robust estimation is needed, e.g.,
in the range of the anticipated BMD or a suspected threshold;

 Consideration of anticipated human exposure levels.

27. The control group shall be an untreated group or a vehicle-control group if a vehicle is used in
administering the test chemical. Except for treatment with the test chemical, animals in the control group
should be handled in an identical manner to those in the test groups. If a vehicle is used, the control
group shall receive the vehicle in the highest volume used among the dose groups. If a test chemical is
administered in the diet, and causes significantly reduced dietary intake due to the reduced palatability of
the diet, an additional pair-fed control group may be useful, to serve as a more suitable control.

Preparation of doses and administration of test chemical

28. The test chemical is normally administered orally, via the diet or drinking water, or by gavage.
Additional information on routes and methods of administration is provided in Guidance Document
No. 116 (7). The route and method of administration is dependent on the purpose of the study, the
physical/chemical properties of the test chemical, its bioavailability and the predominant route and
method of exposure of humans. A rationale should be provided for the chosen route and method of
administration. In the interest of animal welfare, oral gavage should normally be selected only for those
agents, for which this route and method of administration reasonably represent potential human exposure
(e.g., pharmaceuticals). For dietary or environmental chemicals including pesticides, administration is
typically via the diet or drinking water. However, for some scenarios, e.g., occupational exposure,
administration via other routes may be more appropriate.

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29. Where necessary, the test chemical is dissolved or suspended in a suitable vehicle.
Consideration should be given to the following characteristics of the vehicle and other additives, as
appropriate: effects on the absorption, distribution, metabolism, or retention of the test chemical; effects
on the chemical properties of the test chemical which may alter its toxic characteristics; and effects on
the food or water consumption or the nutritional status of the animals. It is recommended that, wherever
possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a
solution/ emulsion in oil (e.g., corn oil) and then by possible solution in other vehicles. For vehicles other
than water, the toxic characteristics of the vehicle should be known. Information should be available on
the stability of the test chemical and the homogeneity of dosing solutions or diets (as appropriate) under
the conditions of administration (e.g., diet).

30. For substances administered via the diet or drinking water it is important to ensure that the
quantities of the test chemical involved do not interfere with normal nutrition or water balance. In long-
term toxicity studies using dietary administration, the concentration of the chemical in the feed should
not normally exceed an upper limit of 5% of the total diet, in order to avoid nutritional imbalances. When
the test chemical is administered in the diet, either a constant dietary concentration (mg/kg diet or ppm)
or a constant dose level in terms of the animal’s body weight (mg/kg body weight), calculated on a
weekly basis, may be used. The alternative used should be specified.

31. In the case of oral administration, the animals are dosed with the test chemical daily (seven
days per week), normally for a period of 24 months for rodents (see also paragraph 32). Any other dosing
regime, e.g., five days per week, needs to be justified. In the case of dermal administration, animals are
normally treated with the test chemical for at least 6 hours per day, 7 days per week, as specified in TG
410 (11), for a period of 24 months. Exposure by the inhalation route is carried out for 6 hours per da , 7
days per week, but exposure for 5 days per week may also be used, if justified. The period of exposure
will normally be for a period of 24 months. If rodent species other than rats are exposed nose-only,
maximum exposure durations may be adjusted to minimise species-specific distress. A rationale should
be provided when using an exposure duration less than 6 hours per day. See also TG 412 (9).

32. When the test chemical is administered by gavage to the animals, this should be done using a
stomach tube or a suitable intubation cannula, at similar times each day. Normally a single dose will be
administered once daily; where for example a compound is a local irritant, it may be possible to maintain
the daily dose-rate by administering it as a split dose (twice a day). The maximum volume of liquid that
can be administered at one time depends on the size of the test animal. The volume should be kept as low
as practical, and should not normally exceed 1 ml/100g body weight for rodents (31). Variability in test
volume should be minimised by adjusting the concentration to ensure a constant volume at all dose
levels. Potentially corrosive or irritant substances are the exception, and need to be diluted to avoid
severe local effects. Testing at concentrations that are likely to be corrosive or irritant to the
gastrointestinal tract should be avoided.

Duration of study
33. The duration of the study will normally be 24 months for rodents, representing the majority of
the normal life span of the animals to be used. Shorter or longer study durations may be used, dependent
on the lifespan of the strain of the animal species in the study, but should be justified. For specific strains
of mice, e.g., AKR/J, C3H/J or C57BL/6J strains a duration of 18 months may be more appropriate. The
following provides some guidance on duration, termination of the study and survival; further guidance,
including consideration of the acceptability of a negative carcinogenicity relative to survival in the study,
is provided in the OECD Guidance Document No. 116 on the Design and Conduct of Chronic Toxicity
and Carcinogenicity Studies(7).

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 Termination of the study should be considered when the number of survivors in the
lower dose groups or the control group falls below 25 per cent.

 In the case where only the high dose group dies prematurely due to toxicity, this should
not trigger termination of the study.

 Survival of each sex should be considered separately.

 The study should not be extended beyond the point when the data available from the
study are no longer sufficient to enable a statistically valid evaluation to be made.

OBSERVATIONS

34. All animals should be checked for morbidity or mortality, usually at the beginning and the end
of each day, including at weekends and holidays. Animals should additionally be checked once a day for
specific signs of toxicological relevance, taking into consideration the peak period of anticipated effects
after dosing in the case of gavage administration. Particular attention should be paid to tumour
development; and the time of tumour onset, location, dimensions, appearance, and progression of each
grossly visible or palpable tumour should be recorded.

Body weight, food/water consumption and food efficiency

35. All animals should be weighed at the start of treatment, at least once a week for the first 13
weeks and at least monthly thereafter. Measurements of food consumption and food efficiency should be
made at least weekly for the first 13 weeks and at least monthly thereafter. Water consumption should be
measured at least weekly for the first 13 weeks and at least monthly thereafter when the substance is
administered in drinking water. Water consumption measurements should also be considered for studies
in which drinking activity is altered.

Haematology, clinical biochemistry and other measurements

36. In order to maximise the information obtained from the study, especially for mode of action
considerations, blood samples may be taken for haematology and clinical biochemistry, and this at the
discretion of the study director. Urinalysis may also be appropriate. Further guidance on the value of
taking such samples as part of a carcinogenicity study is provided in Guidance Document No. 116 (7). If
considered appropriate, blood sampling for haematological and clinical chemistry determinations and
urinalysis may be conducted as part of an interim kill (paragraph 20) and at study termination on a
minimum of 10 animals per sex per group. Blood samples should be taken from a named site, for
example by cardiac puncture or from the retro-orbital sinus under anaesthesia, and stored, if applicable,
under appropriate conditions. Blood smears may also be prepared for examination, particularly if bone
marrow appears to be the target organ, although the value of such examination for the assessment of
carcinogenic/oncogenic potential has been questioned (32).

PATHOLOGY

Gross necropsy
37. All animals in the study except sentinel animals (see paragraph 20) and other satellite animals
should be subjected to a full, detailed gross necropsy which includes careful examination of the external
surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
Sentinel animals and other satellite animals may require necropsy on a case-by-case basis, at the

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discretion of the study director. Organ weights are not normally part of a carcinogenesis study, since
geriatric changes and, at later stages, the development of tumours confounds the usefulness of organ
weight data. They may, however, be critical to performing a weight of evidence evaluation and especially
for mode of action considerations. If they are part of a satellite study, they should be collected at no later
than one year after initiation of the study.

38. The following tissues should be preserved in the most appropriate fixation medium for both the
type of tissue and the intended subsequent histopathological examination (33) (tissues in square brackets
are optional):

all gross lesions heart pancreas stomach (forestomach,

glandular stomach)
adrenal gland ileum parathyroid gland [teeth]
aorta jejunum peripheral nerve testis
brain (including kidney pituitary thymus
sections of cerebrum,
cerebellum, and
medulla/pons)
caecum lacrimal gland prostate thyroid
(exorbital)
cervix liver rectum [tongue]
coagulating gland lung salivary gland trachea
colon lymph nodes (both seminal vesicle urinary bladder
superficial and deep)
duodenum mammary gland skeletal muscle uterus (including
(obligatory for cervix)
females and, if visibly
dissectable, from
males)
epididymis [upper respiratory skin [ureter]
tract, including nose,
turbinates, and
paranasal sinuses]
eye (including retina) oesophagus spinal cord (at three [urethra]
levels: cervical, mid-
thoracic, and lumbar)
[femur with joint] [olfactory bulb] spleen vagina
gall bladder (for ovary [sternum], section of bone
species other than rat) marrow and/or a fresh
bone marrow aspirate
Harderian gland

In the case of paired organs, e.g., kidney, adrenal, both organs should be preserved. The clinical
and other findings may suggest the need to examine additional tissues. Also, any organs
considered likely to be target organs based on the known properties of the test chemical should be
preserved. In studies involving the dermal route of administration, the list of organs as set out for
the oral route should be preserved, and specific sampling and preservation of the skin from the site
of application is essential. In inhalation studies, the list of preserved and examined tissues from the
respiratory tract should follow the recommendations of TG 412 (9) and TG 413 (10). For other
organs/tissues (and in addition to the specifically preserved tissues from the respiratory tract) the
list of organs as set out for the oral route should be examined.

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Histopathology
39. Guidance is available on best practices in the conduct of toxicological pathology studies (33).
The minimum tissues examined should be:

 All tissues from the high dose and control groups;

 All tissues of animals dying or killed during the study;
 All tissues showing macroscopic abnormalities including tumours;
 When treatment-related histopathological changes are observed in the high dose group,
those same tissues are to be examined from all animals in all other dose groups;
 In the case of paired organs, e.g., kidney, adrenal, both organs should be examined.

DATA AND REPORTING

Data
40. Individual animal data should be provided for all parameters evaluated. Additionally, all data
should be summarised in tabular form showing for each test group the number of animals at the start of
the test, the number of animals found dead during the test or killed for humane reasons and the time of
any death or humane kill, the number showing signs of toxicity, a description of the signs of toxicity
observed, including time of onset, duration, and severity of any toxic effects, the number of animals
showing lesions, the type of lesions and the percentage of animals displaying each type of lesion.
Summary data tables should provide the means and standard deviations (for continuous test data) of
animals showing toxic effects or lesions, in addition to the grading of lesions.

41. Historical control data may be valuable in the interpretation of the results of the study, e.g. in
the case when there are indications that the data provided by the concurrent controls are substantially out
of line when compared to recent data from control animals from the same test facility/colony. Historical
control data, if evaluated, should be submitted from the same laboratory and relate to animals of the same
age and strain generated during the five years preceding the study in question.

42. When applicable, numerical results should be evaluated by an appropriate and generally
acceptable statistical method. The statistical methods and the data to be analysed should be selected
during the design of the study (paragraph 9). Selection should make provision for survival adjustments, if
needed.

Test report
43. The test report should include the following information:

Test chemical:
 physical nature, purity, and physicochemical properties;
 identification data;
 source of substance;
 batch number;
 certificate of chemical analysis;

Vehicle (if appropriate):

 justification for choice of vehicle (if other than water);

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Test animals:
 species/strain used and justification for choice made;
 number, age, and sex of animals at start of test;
 source, housing conditions, diet, etc.;
 individual weights of animals at the start of the test;

Test conditions:
 rationale for route of administration and dose selection;
 when applicable, the statistical methods used to analyse the data;
 details of test chemical formulation/diet preparation.
 analytical data on achieved concentration, stability and homogeneity of the preparation;
 route of administration and details of the administration of the test chemical;
 for inhalation studies, whether nose only or whole body;
 actual doses (mg/kg body weight/day), and conversion factor from diet/drinking water
test chemical concentration (mg/kg or ppm) to the actual dose, if applicable;
 details of food and water quality;

Results (summary tabulated data and individual animal data should be presented)

General
 survival data;
 body weight/body weight changes;
 food consumption, calculations of food efficiency, if made, and water consumption,
if applicable;
 toxicokinetic data if available;
 opthalmoscopy (if available);
 haematology (if available);
 clinical chemistry (if available);

Clinical findings
 Signs of toxicity;
 Incidence (and, if scored, severity) of any abnormality;
 Nature, severity, and duration of clinical observations (whether transitory or permanent);

Necropsy data
 Terminal body weight;
 Organ weights and their ratios, if applicable;
 Necropsy findings; Incidence and severity of abnormalities;

Histopathology
 Non neoplastic histopathological findings,;
 Neoplastic histopathological findings;
 Correlation between gross and microscopic findings;
 Detailed description of all treatment-related histopathological findings including
severity gradings;
 Report of any peer review of slides;

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Statistical treatment of results, as appropriate

Discussion of results including

 Discussion of any modelling approaches;
 Dose-response relationships;
 Historical control data;
 Consideration of any mode of action information;
 BMD, NOAEL or LOAEL determination;
 Relevance for humans;

Conclusions

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LITERATURE

1. OECD (1995), Report of the Consultation Meeting on Sub-chronic and Chronic

Toxicity/Carcinogenicity Testing (Rome, 1995), internal working document, Environment
Directorate, OECD, Paris.

2. EPA (2005). Guidelines for Carcinogen Risk Assessment Risk Assessment Forum U.S.
Environmental Protection Agency Washington, DC
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=116283&CFID=1267360&CFTOKEN=65052793&jsessionid=983
0b2c4116e3d8fbbf017414e1a782e7f79TR

3. Combes RD, Gaunt, I, Balls M (2004). A Scientific and Animal Welfare Assessment of the OECD
Health Effects Test Guidelines for the Safety Testing of Chemicals under the European Union
REACH System. ATLA 32, 163-208.

4. Barlow SM, Greig JB, Bridges JW et al (2002). Hazard identification by methods of animal-based
toxicology. Food. Chem. Toxicol. 40, 145-191.

5. Chhabra RS, Bucher JR, Wolfe M, Portier C (2003). Toxicity characterization of environmental
chemicals by the US National Toxicology Programme: an overview. Int. J. Hyg. Environ. Health
206, 437-445.

6. OECD (1998), Repeat Dose 90-day Oral Toxicity Study in Non-Rodents, Test Guideline No. 409,
OECD Guidelines for the Testing of Chemicals, OECD, Paris.

7. OECD (2009), Draft Guidance Document on the Design and Conduct of Chronic Toxicity and
Carcinogenicity Studies, Series on Testing and Assessment No. 116, available on the OECD public
website for Test Guideline at www.oecd.org/env/testguidelines.

8. OECD (2009), Guidance Document on Acute Inhalation Toxicity Testing, Series on Testing and
Assessment No. 39, ENV/JM/MONO(2009)28, OECD, Paris.

9. OECD (2009), Subacute Inhalation Toxicity: 28-Day Study, Test Guideline No. 412, OECD
Guidelines for the Testing of Chemicals, OECD, Paris.

10. OECD (2009), Subchronic Inhalation Toxicity: 90-Day Study, Test Guideline No. 413, OECD
Guidelines for the Testing of Chemicals, OECD, Paris.

11. OECD (1981), Repeated Dose Dermal Toxicity: 21/28-day Study, Test Guideline No. 410, OECD
Guidelines for the Testing of Chemicals, OECD, Paris.

12. Boobis, A.R., Cohen, S.M., Dellarco, V., McGregor, D., Meek, M.E., Vickers, C., Willcocks, D. &
Farland, W. IPCS Framework for analyzing the Relevance of a Cancer Mode of Action for Humans.
Crit. Rev. in Toxicol, (2006) 36:793-801.

13. Cohen, S.M., Meek, M.E., Klaunig, J.E., Patton, D.E., and Fenner-Crisp, P.A. (2003) The human
relevance of information on carcinogenic Modes of Action: An Overview. Crit. Rev. Toxicol.
33:581-589

© OECD 2018
14 │ 451 OECD/OCDE
14. Holsapple, M.P., Pitot, H.C., Cohen, S.N., Boobis, A.R., Klaunig, J.E., Pastoor, T., Dellarco, V.L. &
Dragan, Y.P. (2006) Mode of Action in Relevance of Rodent Liver Tumors to Human Cancer Risk.
Toxicol. Sci. 89:51-56.

15. Meek, E.M., Bucher, J.R., Cohen, S.M., Dellarco, V., Hill, R.N., Lehman-McKemmon, L.D.,
Longfellow, D.G., Pastoor, T., Seed, J. & Patton, D.E. A Framework for Human Relevance analysis
of Information on Carcinogenic Modes of Action. Crit. Rev. Toxicol. (2003). 33:591-653.

16. Carmichael NG, Barton HA, Boobis AR et al (2006). Agricultural Chemical Safety Assessment: A
Multisector Approach to the Modernization of Human Safety Requirements. Critical Reviews in
Toxicology 36, 1-7.

17. Barton HA, Pastoor TP, Baetcke T et al (2006). The Acquisition and Application of Absorption,
Distribution, Metabolism, and Excretion (ADME) Data in Agricultural Chemical Safety
Assessments. Critical Reviews in Toxicology 36, 9-35.

18. Doe JE, Boobis AR, Blacker A et al (2006). A Tiered Approach to Systemic Toxicity Testing for
Agricultural Chemical Safety Assessment. Critical Reviews in Toxicology 36, 37-68.

19. Cooper RL, Lamb JS, Barlow SM et al (2006). A Tiered Approach to Life Stages Testing for
Agricultural Chemical Safety Assessment. Critical Reviews in Toxicology 36, 69-98.

20. OECD (2002), Guidance Notes for Analysis and Evaluation of Chronic Toxicity and Carcinogenicity
Studies, Series on Testing and Assessment No. 35 and Series on Pesticides No. 14,
ENV/JM/MONO(2002)19, OECD, Paris.

21. OECD (2000), Guidance Document on the recognition, assessment, and use of clinical signs as
humane endpoints for experimental animals used in safety evaluation, Series on Testing and
Assessment No. 19, ENV/JM/MONO(2000)7, OECD, Paris.

22. Rhomberg, LR, Baetcke, K, Blancato, J, Bus, J, Cohen, S, Conolly, R, Dixit R, Doe, J, Ekelman, K,
Fenner-Crisp, P, Harvey, P, Hattis, D, Jacobs, A, Jacobson-Kram, D, Lewandowski, T, Liteplo, R,
Pelkonen, O, Rice, J, Somers, D, Turturro, A, West, W, Olin, S. Issues in the Design and
Interpretation of Chronic Toxicity and Carcinogenicity Studies in Rodents: Approaches to Dose
Selection Crit Rev. Toxicol. 37 (9) 729 - 837 (2007).

23. ILSI (International Life Sciences Institute) (1997). Principles for the Selection of Doses in Chronic
Rodent Bioassays. Foran JA (Ed.). ILSI Press, Washington, DC.

24. Griffiths SA, Parkinson C, McAuslane JAN and Lumley CE (1994) The utility of the second rodent
species in the carcinogenicity testing of pharmaceuticals. The Toxicologist 14(1):214.

25. Usui T, Griffiths SA and Lumley CE (1996). The utility of the mouse for the assessment of the
carcinogenic potential of pharmaceuticals. In D'Arcy POF & Harron DWG (eds). Proceedings of the
Third International Conference on Harmonisation. Queen's University Press, Belfast. pp 279-284.

26. Carmichael NG, Enzmann H, Pate I, Waechter, F (1997). The Significance of Mouse Liver Tumor
Formation for Carcinogenic Risk Assessment: Results and Conclusions from a Survey of Ten Years
of Testing by the Agrochemical Industry. Environ Health Perspect 105:1196-1203

© OECD 2018
 OECD/OCDE 451 │ 15

27. EEC Council Directive 86/609/EEC on the approximation of laws, regulations, and administrative
provisions of the Member States regarding the protection of animals used for experimental and other
scientific purposes. Official Journal 29, L358, 18 December 1986.

28. National Research Council, 1985. Guide for the care and use of laboratory animals. NIH Publication
No. 86-23. Washington, D.C., US Dept. of Health and Human Services.

29. GV-SOLAS (Society for Laboratory Animal Science, Gesellschaft für Versuchstierkunde, 1988).
Publication on the Planning and Structure of Animal Facilities for Institutes Performing Animal
Experiments. ISBN 3-906255-04-2. http://www.gv-solas.de/publ/heft1_1988.pdf

30. GV-SOLAS (Society for Laboratory Animal Science, Gesellschaft für Versuchstierkunde, 2006).
Microbiological monitoring of laboratory animals in various housing systems. http://www.gv-
solas.de/auss/hyg/hyg-p7_e.html

31. Diehl K-H, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, Vidal J-M, van de
Vorstenbosch C. 2001. A good practice guide to the administration of substances and removal of
blood, including routes and volumes. Journal of Applied Toxicology, 21:15-23. Available at:
http://www.ff.up.pt/farmacologia/pdf/good_practice_lab_animals.pdf

32. Weingand, K., et al. 1996. Harmonization of Animal Clinical Pathology Testing in Toxicity and
Safety Studies. Fund. Appl. Toxicol. 29: 198-201.

33. Crissman, J., Goodman D., Hildebrandt P., et al. (2004). Best Practices Guideline: Toxicological
Histopathology. Toxicologic Pathology 32, 126-131.

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